Limnol. Oceanogr., 52(5), 2007, 2260–2269
2007, by the American Society of Limnology and Oceanography, Inc.
E
Nitrogen-fixation strategies and Fe requirements in cyanobacteria
Ilana Berman-Frank1
Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan, Israel, 52900
Antonietta Quigg
Department of Marine Biology, Texas A&M University at Galveston, 5007 Avenue U, Galveston, Texas 77551
Zoe V. Finkel
Environmental Science Program, Mount Allison University, Sackville, New Brunswick E4L 1A7, Canada
Andrew J. Irwin
Mathematics and Computer Science Department, Mount Allison University, Sackville, New Brunswick E4L 1E6, Canada
Liti Haramaty
Institute of Marine and Coastal Sciences, Rutgers University, New Brunswick, New Jersey 08901
Abstract
Diazotrophic (nitrogen-fixing) cyanobacteria are important contributors of new nitrogen to oligotrophic
environments and greatly influence oceanic productivity. We investigated how iron availability influences the
physiology of cyanobacterial diazotrophs with different strategies for segregating nitrogen fixation and
photosynthesis. We examined growth, photosynthesis, nitrogen fixation, and Fe requirements of the filamentous
nonheterocystous Trichodesmium, the filamentous heterocystous Anabaena, and the unicellular Cyanothece under
a range of Fe concentrations. Under similar Fe concentrations the three species differed in N2-fixation rates,
photosynthetic activity, the relative abundance of the photosynthetic units PSI : PSII, elemental stoichiometry,
and Fe use efficiency. Complex colonial forms such as Trichodesmium and Anabaena are more likely to be Fe
limited in their natural environments and are more efficient at utilizing Fe than unicellular diazotrophs such as
Cyanothece. The varied physiological responses to Fe availability of the three cyanobacteria reflect their nitrogenfixation strategies, cell size, unicellular or colonial organization, and may explain, at least in part, the ecological
distribution of these photosynthetic bacteria.
Biological fixation of atmospheric nitrogen in the oceans
(primarily by diazotrophic cyanobacteria) is an important
source of ‘‘new’’ nitrogen into the surface oceans that fuels
primary production, phytoplankton blooms, and influences
global carbon cycling (Dugdale and Goering 1967; Falkowski 1997; Karl et al. 1997). Several factors control
aquatic nitrogen fixation. The atmospheric concentration
of oxygen and the corresponding dissolved oxygen in
aquatic systems exert a fundamental control, as nitroge1 Corresponding
author ([email protected]).
Acknowledgments
We thank Kevin Wyman and Paul Field at Rutgers University
for the CHN analysis and for assistance with the ICPMS
respectively. Thanks to John Waterbury at WHOI for cultures
of Trichodesmium and Cyanothece sp. (WH8904). Parts of the
study were performed at the laboratory of P.G. Falkowski, IMCS,
Rutgers University, New Jersey.
Funding sources to A.Q. and L.H. were by NSF OCE 0084032
Biocomplexity: The Evolution and Radiation of Eukaryotic
Phytoplankton Taxa to P. G. Falkowski, Rutgers graduate
fellowship to Z.V.F., and Rutgers postdoctoral fellowship to
I.B.F. Further funding: NSERC discovery grants to Z.V.F. and
A.J.I., Texas Institute of Oceanography for assistance to A.Q.,
and a NATO Science Program Collaboration Linkage grant
NATO EST.MD.CLG 981009 to I.B.-F.
nase, the enzyme responsible for N2 fixation, is irreversibly
damaged by molecular oxygen in vitro (Postgate 1998) and
operates at a fraction of its potential activity in vivo
(Bergman 2001; Gallon 2001; Berman-Frank et al. 2003).
Additional regulation of diazotrophic cyanobacterial populations may be via other factors such as temperature
(Staal et al. 2003b), phosphorus limitation (Wu et al. 2000;
Sanudo-Wilhelmy et al. 2001), iron availability (Paerl et al.
1987; Reuter et al. 1992; Berman-Frank et al. 2001a), or in
some areas colimitation by Fe and P (Mills et al. 2004).
Cyanobacteria are the only organisms that actively
evolve oxygen as a by-product of oxygenic photosynthesis
within the same cell or colony of cells where N2 fixation
occurs. The presence of oxygen triggered biochemical and
morphological adaptations in diazotrophic phototrophs
aimed at limiting the inhibitory effects of oxygen on
nitrogenase (Gallon 1992; Bergman et al. 1997; BermanFrank et al. 2003). Cyanobacterial diazotrophs can
temporally segregate photosynthesis and N2 fixation (fixing
C during the day and N2 at night); they can spatially
separate photosynthesis and N2 fixation; or they can use
a combination of spatial and temporal separation (Adams
2000; Gallon 2001; Berman-Frank et al. 2003) (Fig. 1).
In many unicellular diazotrophs (e.g., Cyanothece,
Croccosphaera) nitrogen is fixed during the night when
2260
Cyanobacteria N2 fixation and Fe
Fig. 1. Schematic representation of the different diazotrophic morphologies, strategies of separation of nitrogen fixation
and photosynthesis, and typical surface area : volume (SA : V)
ratios of the three model organisms used in this study: the
filamentous heterocystous Anabaena, the filamentous nonheterocystous Trichodesmium, and the unicellular Cyanothece. White
coloring indicates the localization of nitrogenase within the cells.
Dark gray coloring indicates actively photosynthesizing vegetative
cells. The graphs in the panels illustrate the relative timing of
photosynthesis (solid black line) and nitrogen fixation (dashed
line) during the diel cycle. Figure is modified from Berman-Frank
et al. (2003).
grown under a light : dark (LD) cycle, or under the
subjective scotophase when grown under continuous light
(Bergman et al. 1997 ). High nitrogenase activity coincides
with high respiration rates; with a phase difference of up to
12 h from the peak of photosynthetic activity (Fig. 1).
Energy and reductants are provided via respiration and use
of photosynthetically fixed carbon while cellular pools such
as Fe and Mo are recycled between day and nighttime
cellular requirements (Tuit et al. 2004).
In heterocystous filamentous cyanobacteria such as
Anabaena, nitrogenase is confined to an anaerobic cell,
the heterocyst, that differentiates completely and irreversibly 12–20 h after combined nitrogen sources are removed
from the medium (Adams 2000). Heterocysts are characterized by a thick membrane that slows the diffusion of O2,
high photosystem I (PSI) activity, the absence of photosystem II (PSII), and loss of division capability. Heterocystous organisms cannot obtain reductant directly from
noncyclic photosynthetic electron flow and rely on the
supply of fixed carbon from adjacent vegetative cells for
reducing equivalents and on cyclic electron flow around
PSI to supply adenosine triphosphate (ATP) (Adams 2000).
A mixed temporal and spatial separation strategy
characterizes the marine nonheterocystous filamentous
cyanobacteria Trichodesmium and Katagnymene (Katagny-
2261
mene has been reaffiliated to Trichodesmium [Lundgren et
al. 2001]). Unlike all other nonheterocystous species of
cyanobacteria, these species fix nitrogen during the day in
a fraction of the cells that are often arranged consecutively
along the trichome (Fig. 1). Active photosynthetic components (such as PSI and PSII complexes, Rubisco, carboxysomes) are found in all cells, even those harboring
nitrogenase (Janson et al. 1994; Fredriksson et al. 1998;
Berman-Frank et al. 2001a). Hence, spatial aggregation of
nitrogenase into certain zones (or cells) is not sufficient to
protect against oxygen. Protection against oxygen in
Trichodesmium is a complex interaction between spatial
and temporal segregation of the photosynthetic, respiratory, and nitrogen-fixation processes (Chen et al. 1999;
Berman-Frank et al. 2001a) with down-regulation of
photosynthetic oxygen evolution and high oxygen consumption through processes such as the Mehler reaction
occurring during the peak of nitrogen fixation (BermanFrank et al. 2001b; Küpper et al. 2004; Milligan et al. in
press) (Fig. 1).
As photosynthesis and N2 fixation both require Fe, it is
likely that the varying morphological and biochemical
strategies used to separate N2 fixation from photosynthesis
will affect the Fe requirements for the different types of
diazotrophs. Availability of iron influences N2 fixation in
cyanobacteria by its direct effect on Fe-rich protein
synthesis of nitrogenase, and by effects on photosynthesis,
growth, and global productivity (Paerl et al. 1987;
Falkowski 1997). Higher intracellular iron quotas were
calculated and found in diazotrophic cyanobacteria in
comparison with nondiazotrophic phytoplankton (Kustka
et al. 2003; Tuit et al. 2004). Several studies, mostly focused
on the nonheterocystous filamentous Trichodesmium as the
model organism, have shown a positive correlation between
Fe availability and nitrogen fixation and growth (Reuter et
al. 1992; Paerl et al. 1994; Berman-Frank et al. 2001a).
However, differences in Fe requirements and use by
diazotrophs with different morphological and biochemical
adaptations for segregation of N2 fixation and photosynethesis have not yet been determined.
In this study, we focused on how iron availability
influences cyanobacterial diazotrophs with different strategies for nitrogen fixation: the filamentous nonheterocystous Trichodesmium, the filamentous heterocystous Anabaena, and the unicellular Cyanothece (Fig. 1). We tested
whether the biochemical and morphological differences
(such as cell and colony size) characterizing the spatial or
temporal (or both) segregation of N 2 fixation and
photosynthesis affect the utilization of Fe as reflected in
rates of nitrogen fixation, photosynthesis, and growth over
a range of Fe concentrations.
Materials and methods
Culture growth conditions—Cultures of Trichodesmium
IMS101, Cyanothece sp. (WH8904), and Anabaena flos
aqua (UTEX 2557) were grown at 26uC under a 12 : 12 LD
cycle at 85 mmol m22 s21 quanta supplied by VHO
fluorescent tubes. Trichodesmium data were obtained from
Berman-Frank et al. (2001a) and additional unpublished
2262
Berman-Frank et al.
data from associated experiments. Trichodesmium was
grown in YBCII media (Chen et al. 1996) amended as
below for Fe and ethylenediaminetetra-acetic acid (EDTA).
Cyanothece sp. cultures were grown in a modified Aquil
seawater media prepared as described in Ho et al. (2003).
Anabaena was grown in an artificial freshwater media,
Fraquil, prepared as described by Morel et al. (1975) except
that the trace-metal stock, nutrients (N and P), and
vitamins for the modified Aquil recipe were used rather
than those described for Fraquil. For cultures of all three
species, Fe (FeCl3) was added at five concentrations
between 4 nmol L21 and 4 mmol L21, and complexed with
20 mmol L21 EDTA (see Berman-Frank et al. 2001a for
details). The media was microwave-sterilized at least 24 h
before use. Cyanothece and Anabaena were grown in each
Fe treatment for a minimum of five generations before the
commencement of experiments to fully acclimate cells to
their respective Fe treatments. Cultures were kept optically
thin throughout the experiment to prevent physiological
changes due to decreased irradiance rather than Fe effects.
Unless noted otherwise, cells were harvested for experiments during the exponential phase of growth. Although
cultures were not axenic, extremely low bacterial counts
were observed during the exponential phase. Growth rates
were determined from linear regression analysis of the
increase of logarithmic transformed C and N concentrations versus time. CHN was assayed on precombusted
Gelman AE filters using a Carlo-Erba CHN analyzer.
Cultures were filtered under low vacuum (,13 kPa) and
stored frozen until CHN analysis could be performed.
Chlorophyll a—Samples for chlorophyll a (Chl a)
analysis were filtered gently onto 13-mm 0.45-mm Durapore
(Millipore) filters and frozen at 220uC. Filters were
defrosted slowly on ice in dim light. Methanol (90%) was
added and the samples were sonicated with a Branson
sonicator while on ice. Samples were returned to the freezer
overnight and centrifuged the next day to clarify. Chl
a concentrations were assayed at 664 and 750 nm using
a HP 8451A diode array spectrophotometer. At least two
samples were taken for each of the three cultures of
Cyanothece and Anabaena grown in each Fe treatment and
replicate results were averaged.
Metal quotas—Inductively coupled mass spectrometry
(ICPMS) measurements were made essentially as described
previously (Berman-Frank et al. 2001a ; Quigg et al. 2003)
with the following modifications for Anabaena and
Cyanothece. Samples were collected onto 25-mm Poretics
filters that had been pretreated as described in Cullen and
Sherrell (1999) on a filter apparatus detailed in Ho et al.
2003 under gentle filteration (,13 kPa). All filtering was
done in a laminar flow hood to reduce contamination of
samples by airborne particles. Additional filters were
washed with Aquil or Fraquil and 0.4 mol L21 NaCl to
assay background contamination. Trichodesmium was
examined as described in Berman-Frank et al. (2001a).
Nitrogenase activity—Nitrogen-fixation rates for all
three species were measured using the acetylene reduction
method according to Capone (1993). Ten to 30 mL of
cultures (depending on biomass) were sealed in serum
bottles, injected with acetylene (20% of headspace volume),
and incubated for 2 h at ambient light and temperature.
Ethylene production was measured on a SRI 310 gas
chromatograph with a flame ionization detector and
quantified relative to an ethylene standard. Results were
normalized to carbon and nitrogen.
Variable fluorescence and photosynthetic parameters—
We used a fast repetition rate fluorometer, which measures
fluorescence transients induced by a series of subsaturating
excitation pulses from a bank of blue-green light-emitting
diodes to derive photosynthetic parameters (Kolber et al.
1998). The photochemical quantum yield (Fv : Fm) was
determined from the initial, dark-adapted fluorescence (Fo)
and the maximal fluorescence (Fm) when all PSII reaction
centers are photochemically reduced (Fv : Fm 5 (Fm 2 Fo)/
Fm). Blanks of sterile media were subtracted from each
measurement. The functional absorption cross-section of
photosystem II (sPSII) was calculated according to Kolber
et al. (1998).
Relative abundance of PSI and PSII—The ratio between
PSI and PSII was estimated from 77K fluorescence
emission spectra (excitation wavelength 435 nm) performed
on cells that were flash-frozen in liquid nitrogen. The
spectra were analyzed on an AB2 Aminco spectrofluorometer equipped with a liquid nitrogen Dewar, which allowed
measurements at 77K using a 2-nm bandpass. Spectra were
resolved, machine baseline subtracted, and the peak ratio
was determined using PeakFitTM Software (SPSS). Spectra
were normalized to emission at 685 nm.
Surface area and volume parameters—Biovolume and
surface area were calculated using Biovol2.1 software
(courtesy David Kirschtel) assuming cylinder shapes for
Trichodesmium (filament lengths 100 to 1000 mm, cellular
radius 4 mm) and Anabaena (filament lengths 100 to
1000 mm, cellular radius 1.3 mm) and a sphere (average
radius 1.4 mm) for Cyanothece.
Results
The effects of iron on growth, photosynthesis, the
relative ratios of PS I and II, cellular iron quotas, and
nitrogen-fixation rates were measured in exponential-phase
cultures of Anabaena and Cyanothece. We include some
new data and also findings from earlier work (BermanFrank et al. 2001a) on Trichodesmium for reference since it
was grown under identical conditions to the marine
diazotroph, Cyanothece.
Growth rates—All cyanobacteria were acclimated and
grown under varying iron regimes (range 4 nmol L21 to
4 mmol L21) for a minimum of five generations. Of the
three species tested, carbon-specific growth rates were
highest in the unicellular Cyanothece at all Fe concentrations (Table 1, Fig. 2). An increase in iron concentrations
in the growth media of Cyanothece cultures resulted in
Cyanobacteria N2 fixation and Fe
2263
Table 1. The influence of Fe availability on growth rates and elemental ratios of Fe to C and P in Trichodesmium, Cyanothece, and
Anabaena. Values are averages of n53 for Anabaena and Cyanothece, n54 for Trichodesmium at all Fe concentrations except
Fe54 mmol L21 where n512. Numbers in parentheses represent standard errors.
External Fe concentration ( mmol L21)
Carbon-based growth, m
Anabaena
Trichodesmium
Cyanothece
Fe : P (mmol : mol)
Anabaena
Trichodesmium
Cyanothece
FPUE (mmol Fe mol P21 h21)
Anabaena
Trichodesmium
Cyanothece
Fe : C (mmol : mol)
Anabaena
Trichodesmium
Cyanothece
FUE (mmol Fe mol C21 h21) 3 1023
Anabaena
Trichodesmium
Cyanothece
0.004
0.04
0.1
0.4
4.0
0.14 (0.003)
0.05 (0.004)
0.23 (0.02)
0.15 (0.009)
0.06 (0.007)
0.26 (0.005)
0.23 (0.03)
0.09 (0.008)
0.29 (0.018 )
0.26 (0.02)
0.1 (0.009)
0.31 (0.05)
0.26 (0.02)
0.12 (0.01)
0.28 (0.04)
4.2 (3)
0.8 (0.006)
6 (2)
5.6 (1)
1.6 (0.8)
7.3 (1)
2.4 (1)
2 (0.1)
26 (3)
5.4 (2)
3 (0.5)
40 (2)
30 (7)
22 (7)
271 (22)
0.04
0.004
0.09
0.023
0.007
0.31
(d21)
0.025
0.002
0.06
0.076 (0.05)
13 (9.9)
0.076 (0.014)
0.32 (0.012)
30 (2.7)
0.074 (0.014)
0.4
27
0.73
2
75
0.8
a modest increase of up to 36% for the C-specific growth
rates (Fig. 2). Both Anabaena and Trichodesmium responded to increases in external Fe with a sigmoidal-type
growth curve. The lowest growth rates occurred at 4 nmol
L21 to 0.04 mmol L21, and increased linearly until Fe
concentrations reached 0.4 mmol L21. Fe concentrations
greater than ,1 mmol L21 did not result in higher growth
rates. Both Cyanothece and Anabaena grew significantly
faster than Trichodesmium at the same Fe concentrations;
with division rates at 0.25, 0.27, and 0.12 d21 respectively
at 4 mmol L21 Fe (Fig. 2).
Photosynthetic performance—The photochemical quantum yield of PS II photochemistry, Fv : Fm, provides an
early diagnostic for iron limitation (Kolber et al. 1994;
Behrenfeld and Kolber 1999). Photochemical quantum
yields were positively correlated with carbon-specific
growth rates for the three species with a saturation
response of Fv : Fm observed at high Fe concentrations
(.0.1 mmol L21) for the three species examined (Fig. 3).
The unicellular Cyanothece outperformed the two filamentous species and maintained high photosynthetic efficiency
as reflected in the maximum quantum yields of PSII, which
increased from 0.5 to 0.78 over a range from 4 nmol L21 to
4 mmol L21 Fe (Fig. 3). Anabaena responded to enhanced
Fe availability by a linear increase in Fv : Fm from values of
,0.35 at 4 nmol L21 Fe to values ,0.6 as Fe availability
increased to 0.1 mmol L21 (Fig. 3). The slight decline in
Fv : Fm observed for Anabaena at 4 mmol L21 Fe is not
significant and falls within the range of saturated values .
Trichodesmium showed a response similar to the other two
cyanobacteria examined but maintained consistently lower
photosynthetic efficiencies. Photochemical yields, Fv : Fm,
0.16 (0.02)
33 (1.8)
0.37 (0.08)
1.5
124
4.5
0.06
0.01
0.52
0.2 (0.018)
48 (2.2)
0.58 (0.07)
2.2
194
7.5
0.32
0.11
3.16
2.4 (0.3)
168 (23)
9.38 (2.4)
26
840
109
determined at the peak of nitrogenase activity (i.e., about
5 h from light induction) were 50% lower (Fv : Fm 5 0.25)
for iron-depleted cultures at 4 nmol L21 Fe compared with
iron-replete cultures (Fv : Fm 5 0.54 at 4 mmol L21 Fe)
(Fig. 3).
Fig. 2. (A) Effect of Fe availability on carbon-specific
growth rates mc (d21) in Trichodesmium, Cyanothece, and
Anabaena.
2264
Berman-Frank et al.
Fig. 3. Influence of Fe availability on photochemical quantum yields of PSII (Fv : Fm) in exponentially growing cultures of
Trichodesmium, Cyanothece, and Anabaena.
Stoichiometry of photosynthetic units—The relative
abundances of PSI and PSII vary in diazotrophs because
of the additional roles that the two photosystems play in
the nitrogen-fixation process, and because of their high
atomic requirement. For all three species, emission spectra
at 77K showed distinct changes in the spectral signature as
Fe availability changed (Fig. 4A–C). In general, Fe-depleted cultures displayed spectra with a more dominant
PSII component compared with PSI (Fig. 4 A–C), sometimes resulting in both the 685-nm and 695-nm peaks
representing the core antennae complexes of PSII, CP43
and CP47, respectively. Both Trichodesmium and Anabaena
exhibited much more prominent PSI signatures at high Fe
(Fig. 4A,B) than Cyanothece (Fig. 4C), in which PSII was
always dominant.
In Trichodesmium IMS101, Fe availability influenced the
relative abundance of PSI and PSII. Peak area ratios of
PSI : PSII decreased threefold, from 1.3 6 0.3 to 0.25 6
0.06 as Fe was reduced from 4 mmol L21 to 4 nmol L21
(Figs. 4A, 5A). The changes in PSI : PSII in Trichodesmium
were correlated with Fe availability (Fig. 5A) and with
nitrogen-fixation rates (r2 5 0.98) (Fig. 5B). In Cyanothece
PSI : PSII ratios increased with Fe availability (Fig. 5A),
but no correlation was observed between PSI : PSII ratios
and nitrogen-fixation rates for this unicellular organism
(Fig. 5B). In the heterocystous Anabaena, PSI : PSII ratios
were consistently higher at all Fe concentrations (Fig. 5A)
with two levels of ratios measured: PSI : PSII , 1.5 at the
two lowest Fe concentrations, then significantly higher
ratios (at Fe . 0.1 mmol L21) ranging from 4.5 to 6.5.
(Fig. 5A). In Anabaena, no change in the PSI : PSII was
observed for all but the lowest nitrogen-fixation rates
(when Fe is limited) (Fig. 5B).
Fig. 4. The influence of Fe availability on the 77K fluorescence emission spectra and PSI : PSII for whole cells of (A)
Trichodesmium, (B) Anabaena, and (C) Cyanothece. Spectra are
representative of the highest Fe concentration (4 mmol L21, solid
lines) and the lowest concentrations (4 nmol L21, dashed lines).
Excitation wavelength is 435 nm. Spectra were normalized to
peaks at 687 nm (PSII).
Nitrogen fixation—Nitrogen-fixation rates in Trichodesmium were significantly affected by iron availability with
a sigmoidal pattern observed over the range of Fe
concentrations in this study (Fig. 6). When Fe was reduced
from 4 to 0.1 mmol L21, rates of ethylene produced
decreased by 50% and by more than 85% when Fe
availability was reduced to 4 nmol L21 , with nitrogenfixation rates declining sevenfold from 0.5 to 0.07 mmol N
mol C21 h21 (Fig. 6). Anabaena and Cyanothece did not
Cyanobacteria N2 fixation and Fe
2265
Fig. 6. Influence of Fe availability on nitrogen fixation rates
in exponentially growing cultures of Trichodesmium, Cyanothece,
and Anabaena.
Fig. 5. (A) Influence of Fe availability on PSI : PSII ratios,
and (B) relation between PSI : PSII ratios and nitrogen-fixation
rates in Trichodesmium, Cyanothece, and Anabaena.
show the same dependence on Fe availability over the same
concentration range as Trichodesmium (Fig. 6). In Anabaena, only the lowest Fe concentration (4 nmol L21)
showed any significant reduction (about 50% from maximal values) in nitrogen-fixation rates. At all other Fe
concentrations nitrogen-fixation rates (normalized to carbon) were saturated and not significantly different from one
another. In Cyanothece similar rates of nitrogen fixation
(1.6–1.8 mmol N mol C21 h21) were observed across the
three-orders-of-magnitude changes in Fe concentrations
and were only 3.5-fold higher than measured rates of
nitrogen fixation in Fe-replete Trichodesmium (Fig. 6).
Fe utilization—The effects of cellular size and resource
utilization were reflected in the Fe : C and Fe : P ratios,
which also varied with Fe availability for all three species
(Table 1). Trichodesmium with the lowest surface area-tovolume ratio (SA : V) and largest-size filaments displayed
much higher intracellular quotas than either Anabaena or
the unicellular small Cyanothece with the largest SA : V.
(Fe : C of Trichodesmium ranged from 13 to 168; for
Anabaena 0.08 to 2.4; or the small unicellular Cyanothece
0.08 to 9.4 from 0.004 to 4 mmol L21 Fe respectively)
(Table 1). These differences in Fe : C quotas were also
reflected in the iron use efficiency (FUE, Fe [C h] 21),
which quantifies the amount of Fe utilized per carbon
relative to time. To avoid the potential bias of extracellular
accumulation of Fe, the FUE is best determined under low
Fe. Trichodesmium with the largest quotas of Fe : C also
had FUE ,27–75, higher than both Anabaena and
Cyanothece (Table 1). In contrast, the Fe : P ratios were
the lowest in Trichodesmium, ranging from 0.8 to 22
compared with Anabaena (4.2 to 22) and Cyanothece (6 to
271) at 0.004 to 4 mmol L21 Fe respectively, with highest
values possibly due to extracellular Fe accumulation. The
low Fe : P of Trichodesmium corresponded to a very
inefficient utilization of Fe per P relative to time (FPUE,
Fe [P h]21) in comparison with both Anabaena and
Cyanothece. The smallest Cyanothece with the largest
SA : V also had a 30-fold higher FPUE, Fe (P h)21 than
Trichodesmium. These results may reflect issues of luxury
consumption of P and extracellular P absorption (see
Discussion).
Discussion
Anabaena, Cyanothece, and Trichodesmium use different
strategies to balance demands of photosynthesis and
2266
Berman-Frank et al.
nitrogen fixation that can be characterized as spatial,
temporal, or a combination of spatial and temporal, along
with use of specialized cells (Fig. 1). In phototrophic
diazotrophs, nitrogen fixation, photosynthesis, and respiration are intricately coupled, with both photosynthesis
and respiration providing ATP and reductant for the
energy-expensive process of nitrogen fixation. Respiration
provides the substrates required for the assimilation of the
fixed nitrogen and for consuming the oxygen derived from
photosynthesis that irreversibly inhibits nitrogenase (Postgate 1998). The evolutionary selection of strategy by
Anabaena, Cyanothece, and Trichodesmium to separate N2
fixation from oxygenic photosynthesis and related morphological and biochemical adaptations have consequences
for nutrient demand, metabolic performance, and preferred
habitat. Growth rates for Cyanothece showed only a very
modest decrease for the two lower Fe concentrations and
overall do not differ significantly across the range of Fe
assayed. In contrast, Trichodesmium and Anabaena exhibited a sigmoidal response as a function of Fe availability,
indicating that these species were exposed to a range of
conditions from Fe-limiting to Fe-replete in our experiments. Although the differential responses in PSI : PSII, N2
fixation, and growth rates may be due to differences in
metabolic strategies, covariations in cell size and colonialism indicate there are likely trade-offs related to Fe or other
nutrient requirements, maximum growth rates, and the
effectiveness of protecting nitrogenase from oxygen.
Temporal segregation of N2 fixation and photosynthesis
into dark and light phases of the day (as in Cyanothece sp.)
is a common mechanism used by unicellular diazotrophs to
separate oxygen evolution of PSII from the O2-sensitive
nitrogenase complex. In this way energy and substrates are
generated during the light hours to fuel the energetically
expensive process of nitrogen fixation, thus reducing the
costs involved in protection of nitrogenase against photosynthetically produced oxygen. Moreover, elements such as
Fe and Mo can be recycled between different cellular
processes such as photosynthesis and nitrogen fixation and
the cells can maintain low cellular quotas. Diel cycling of
Fe : C has been documented in unicellular Croccosphaera,
similar in size (,2 mm) and N2-fixation strategy to
Cyanothece (Tuit et al. 2004). In Croccosphaera higher
Fe : C was measured at night (N2-fixing period) and low
Fe : C ratios during the photoperiod. Ratios of Fe : C
during the nitrogen-fixing period ranged from ,7 to
27 mmol mol21 (Tuit et al. 2004), similar to the values we
measured in Fe-replete cultures of Cyanothece. These
investigators calculated that an average internal concentration of 19–1,000 mmol L21 Fe in Croccosphaera at
abundances of 106 cells L21 would require only 2.4 pmol
L21 Fe compared with 0.1 nmol L21 available in seawater,
indicating that Fe limitation in this organism in the ocean
was unlikely (Tuit et al. 2004). The same appears to be true
for Cyanothece under our experimental conditions. Cyanothece had a high quantum yield for photosynthesis
(Fv : Fm) at all external levels of Fe, with only a modest
decrease in photosynthetic efficiency at the lowest Fe
concentration (Fig. 3). This translated into growth rates
and nitrogen-fixation rates in Cyanothece that were almost
independent of Fe over the three orders of magnitude tested
here, contrasting with the Fe dependence exhibited by the
two other diazotrophs (Fig. 6). Under Fe-replete conditions, Cyanothece exhibited an increase in Fe : P and Fe : C
that may reflect a combination of higher efficiency for P
uptake (Table 1) and decreased energy costs associated
with replete Fe conditions. The apparent insensitivity of
nitrogen fixation in Cyanothece to low Fe concentrations
(Fig. 6), combined with its low iron use efficiency, further
supports the suggestion that small diazotrophs such as
Cyanothece are unlikely to be strongly growth limited by Fe
even at nanomolar concentrations.
The effect of cell size on cellular Fe to C noted by Sunda
and Huntsman (1997) was confirmed in our results (Table 1,
Fig. 1): Trichodesmium (SA : V , 0.5) displayed the highest
Fe : C and both Anabaena (SA : V , 1.5) and Cyanothece
(SA : V ,2) had extremely low Fe : C (Table 1). These
characteristics may allow Cyanothece and other small
unicellular diazotrophs to flourish in traditional Fe-limited
areas of the oceans (Falcon et al. 2004; Langlois et al. 2005)
as long as all other environmental parameters are favorable.
Although the small size and large SA : V of Cyanothece
provides an advantage for Fe and other nutrient uptake, the
potential of the cells for luxury uptake, storage of nutrients,
or spatial division of cellular processes is restricted.
Spatial segregation of N2 fixation is advantageous
because the photosynthate required for the energetically
costly N2 fixation need not be stored until the dark phase but
can be generated and used in the light. Anabaena, known for
its high growth rates (Denobel et al. 1997), generally
maintained growth rates that were ,two- to fourfold greater
than those measured for Trichodesmium over a 1,000-fold
range in ambient Fe concentration (Fig. 2).
Although both Trichodesmium and Anabaena fix nitrogen during the day, in Anabaena the process occurs in the
specialized heterocysts that allow nitrogen fixation to
proceed throughout the photoperiod. Thus N-fixation rates
of Anabaena were higher at all Fe concentrations than
those of Trichodesmium (Fig. 6). This strategy requires
colonial organization and implies intercellular transportation costs (e.g., transportation of fixed N to vegetative cells)
that reduce overall growth rates relative to smaller cells
such as Cyanothece. Many metabolic processes in colonial
organisms are still regulated at the cell level, so the
‘‘effective organism size’’ varies from that of a single cell
to the whole colony, depending on the process.
The major disadvantage of spatially segregating oxygenic photosynthesis and nitrogen fixation arises under Felimiting conditions. In this case, Fe demand is much higher
than in strategies of temporal segregation because of the
need for concomitant N2 fixation and photosynthesis and
the higher PSI : PSII ratios (Fig. 4). It has generally been
assumed that the primary role of PSI in cyanobacteria is to
provide ATP via cyclic electron flow around the reaction
center (Wolk et al. 1994). However, the photocatalyzed
reduction of O2 via PSI (Mehler reaction), which also
generates ATP, is potentially a major sink for both
photosynthetically generated and ambient O2 (BermanFrank et al. 2001b; Milligan et al. in press). Heterocysts
lack the water-splitting, oxygen-evolving PSII and contain
Cyanobacteria N2 fixation and Fe
only PSI complexes that supply ATP and reduce O2. The
two photosystems vary in their Fe demand as PSII is
associated with only three iron atoms (two in cytochrome
b559 and one nonheme iron) and coordinates the two core
reaction center proteins, whereas PSI contains at least 12
iron atoms and is a major sink for iron in all oxygenic
photoautotrophs (Falkowski and Raven 1997).
The importance of PSI for heterocystous diazotrophs
was expressed as a large increase in PSI : PSII in Anabaena
relative to Trichodesmium or Cyanothece at all iron
concentrations, even under very low Fe concentrations
(PSI : PSII . 1 in Anabaena, Fig. 4B). Additionally,
PSI : PSII ratios correlated positively with Fe availability.
When Fe was limiting, PSI declined in relative abundance
and a spectral shift to PSII was observed (Fig. 4A–C). This
was probably due to the induction (or enhancement) of the
isiA gene, which encodes the chlorophyll binding protein
CP439 and may work as an auxiliary PSI antenna (Xu et al.
2003) as reflected in the 77K emission spectra (Fig. 4).
Heterocystous cyanobacteria are predominantly terrestrial, freshwater, and coastal species, inhabiting eutrophic
or brackish environments with a few epiphytic and
symbiotic representatives in the marine environment (Paerl
and Zehr 2000). Very few heterocystous free-living species
are found in the pelagic oceans (Carpenter and Janson
2001). The heterocyst glycolipid envelope appears to confer
a competitive advantage in low-temperature brackish and
freshwater by protecting against the increased O2 flux
compared with that in seawater (Staal et al. 2003a). At
higher temperatures, typical of tropical waters, the
glycolipid envelope of the heterocyst does not provide
additional protection against oxygen, which may explain
the dearth of heterocystous free-living cyanobacteria in
such environments (Staal et al. 2003a). Anabaena grows
mostly in freshwater and brackish environments (Baltic
Sea) where Fe concentrations are at least an order of
magnitude higher than in the oligotrophic oceans. The
higher availability of Fe allows for increased PSI in the
heterocysts and thus high PSI : PSII at all but the very
lowest Fe concentrations (Fig. 5A). This enables concurrent high rates of nitrogen fixation and photosynthesis
yielding high growth rates that more than compensate for
the nonphotosynthesizing heterocyst biomass (Figs. 2, 3,
6). When Fe is limiting, which may occur at times in the
Baltic Sea (Sikorowicz et al. 2005), the lower efficiency in
utilizing Fe results in declining nitrogen fixation (Fig. 6)
and growth rates in Anabaena sp. (Fig. 2).
The combined spatial and temporal segregation strategy
of Trichodesmium is intermediate between the strategies
exhibited by Cyanothece and Anabaena. The lack of
heterocysts requires fine-tuning of photosynthesis and
nitrogen fixation during the photoperiod, limiting the
hours of nitrogen fixation and the amount of energy and
substrates produced by photosynthesis. Photosynthesis
and N2 fixation both occur in the light, but are necessarily
spatially separated. Limited spatial separation occurs
with nitrogen fixation seemingly concentrated in ‘‘diazocytes’’ (Lin et al. 1998; Berman-Frank et al. 2001b).
Incomplete spatial segregation necessitates temporal separation during hours of peak nitrogen fixation with the
2267
down-regulation of PSII, up-regulation of the Mehler
reaction, and other oxygen consumption pathways (Berman-Frank et al. 2001b). The co-occurring demands for
both photosynthesis and nitrogen fixation in Trichodesmium dictate a high degree of efficiency and regulation of
Fe uptake at all external Fe concentrations. Trichodesmium, which inhabits some oceanic areas that are extremely
poor in Fe or P (or both), has a much higher iron use
efficiency than either Anabaena or Cyanothece (Table 1).
When ambient Fe is not limiting, Trichodesmium can
increase Fe uptake and store the excess Fe (Tuit et al.
2004). Trichodesmium may also utilize natural colloidal Fe
although the actual bioavailability of Fe is dependent on
the colloidal chemical characteristics (Wang and Dei 2003).
The finding by Tuit et al. (2004) that, in the absence of
a source of fixed N, the nitrogenase complex in Trichodesmium is more metal (Fe) efficient, is confirmed by our
observation of high iron use efficiency under low iron
concentrations.
Another characteristic providing enhanced plasticity for
Trichodesmium (despite its inherent disadvantage of small
SA : V and low growth rates) is luxury uptake and storage
of P (Krauk et al. 2006; White et al. 2006). Additionally,
Trichodesmium’s ability to utilize phosphonates from the
dissolved organic phosphorus pool (Dyhrman et al. 2006)
and surface-absorbed phosphate (Sanudo-Wilhelmy et al.
2004) may account for the relatively low Fe : P and FPUE
we calculated for this cyanobacterium (Table 1). The
plasticity in P utilization may thus enable higher rates of
N fixation when Fe is replete. Thus at high Fe concentrations N-fixation rates of Trichodesmium were only 3–3.5fold lower than those of the other two diazotrophs tested
here (Fig. 6).
Ancestral nitrogen fixers developed a variety of strategies for coping with their ‘‘oxygen problem.’’ These
included a range of temporal and spatial structuring of
metabolic processes within unicellular and colonial organizations. The morphological and biochemical adaptations
of diazotrophs to oxygen impose a suite of associated
physiological constraints that lead to specific trade-offs
under different aquatic environments. The varied physiological responses to Fe availability of the three diazotrophs
in this study reflect a combination of their nitrogen-fixation
strategies, cell size, and unicellular or colonial organization,
and may explain, at least in part, the ecological distribution
of these organisms.
Unicellular cyanobacterial diazotrophs were traditionally thought to be insignificant in global N fixation, but their
contribution may be greatly underestimated (Zehr et al.
2001; Falcon et al. 2004; Langlois et al. 2005). Their small
size and rapid intracellular Fe recycling capacity makes
unicellular diazotrophs relatively resistant to low concentrations of Fe and P, giving them an advantage in stable,
low-nutrient waters. Colonial organization in diazotrophic
cyanobacteria (e.g., Trichodesmium) permits a temporal
protection of nitrogenase from photosynthetically evolved
oxygen and the simultaneous fixation of nitrogen and
carbon during the day but leads to an increase in cell size
and decrease in SA : V, resulting in a decrease in metabolic
rates such as growth.
2268
Berman-Frank et al.
Heterocystous cyanobacteria require a high investment
of both energy and nutrients for cellular differentiation and
may not be able to grow successfully in high-temperature
and low-Fe and -P environments that characterize large
oceanic regions. In such circumstances either the small
unicellular forms such as Cyanothece or large partially
spatially structured colonial forms such as Trichodesmium
are better adapted. Current estimates suggest that Trichodesmium may be two- to threefold more abundant than
previously reported and may account for the missing sink
of ,90 Tg of N required to support observed oceanic new
production (Davis and McGillicuddy 2006; Kolber 2006).
The relative success of Trichodesmium in oceanic environments may be due to a complex combination of spatial and
temporal separation of nitrogen and carbon fixation, high
iron use efficiency, gas vacuoles, and plasticity in Fe and P
storage that allow this organism to successfully exploit
pulses of nutrients from dust events (Lenes et al. 2001) or
from depth. Low grazing pressures (O’Neil 1998) or even
the presence of an autocatalytic programmed cell death
pathway in Trichodesmium that could select for more
resistant cells (Berman-Frank et al. 2004) may act to
further enhance survival of these larger (colonial bloomforming) forms although their growth and nutrient-uptake
rates are lower compared with small unicellular forms such
as Cyanothece.
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Received: 25 November 2006
Accepted: 2 April 2007
Amended: 17 April 2007